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Abstract

Dopamine is an important catecholamine neurotransmitter modulating many physiological
functions, and is linked to psychopathology of many diseases such as schizophrenia
and drug addiction. Dopamine D1 and D2 receptors are the most abundant dopaminergic
receptors in the striatum, and although a clear segregation between the pathways expressing
these two receptors has been reported in certain subregions, the presence of D1-D2
receptor heteromers within a unique subset of neurons, forming a novel signaling transducing
functional entity has been shown. Recently, significant progress has been made in
elucidating the signaling pathways activated by the D1-D2 receptor heteromer and their
potential physiological relevance.

Background

Dopamine plays a key role in the regulation of various physiological functions of
normal brain including reward, locomotion, behavior, learning, and emotion. It is
not then surprising that the dysregulation of the dopaminergic system has been linked
to pathophysiology of many diseases, such as Alzheimer's disease, schizophrenia, Parkinson's
disease, attention deficit hyperactivity disorder, depression and drug addiction [1-3], leading to the clinical use of drugs that target dopamine neurotransmission in the
treatment of these disorders.

Five subtypes of dopamine receptors (D1R-D5R), belonging to the G-protein-coupled
receptor (GPCR) superfamily have been cloned, through which dopamine transduces its
various effects. Dopamine receptors are subdivided into D1-like (D1, D5) and D2-like
(D2, D3, D4) receptor subclasses [1-3], with the D1 and D2 receptors being the major subtypes. The most studied dopamine
signaling pathway is the modulation of cyclic AMP production, with D1-like receptors
activating cyclic AMP production through Gs/olf, and D2-like receptors inhibiting
adenylyl cyclase (AC) activity through Gi/o proteins [2]. This results in a bidirectional modulation of this pathway and related proteins,
such as protein kinase A (PKA) and DARPP-32 (dopamine and cAMP regulated protein)
[4]. Other important dopamine signaling pathways have also been reported, including the
modulation of the Akt-GSK3 pathway [5] and the activation of the PAR4 signaling pathway [6].

For some actions of dopamine, such as the control of motor behavior [7] or dopamine-mediated reward processes in nucleus accumbens [8], a concomitant stimulation of D1 and D2 receptors is required, a phenomenon known
as the "requisite" D1/D2 synergism [9]. In this type of synergism, D1 and D2 receptor-specific drugs potentiate the effect
exerted by each other when delivered together, but are ineffective when administered
separately [9]. The combined, but not separate, administration of a selective D1 and a selective
D2 agonist was shown to be necessary for the dopamine-stimulated expression of immediate-early
gene c-fos in striatal neurons [10] and in electro-physiological studies where both receptors were indeed responsible
for GABA release in striatum [11]. The participation of both D1 and D2 receptors was also required for evoking neural
and behavioral sensitization to cocaine [12] and for evoking the changes in behavior and basal ganglia output [13,14]. All these observations are other evidence for the presence of not only a synergism
between dopamine D1 and D2 receptors, but an obligatory participation of both receptors
to generate this synergism.

One explanation for how the well documented synergistic effects seen between D1 and
D2 receptors [15,16] may be achieved is through the formation of heterooligomers between the two receptors,
as it has been shown for many GPCRs [17-19]. Dopamine receptors, all subtypes included, in addition to their ability to exist
as homomers, were shown to form different heteromeric complexes with other receptors
(reviewed in 20). The presence of D1-D2 receptor heteromers with unique functional
properties was first shown in transfected cells using different methods [21-24] as described below. Initially, the notion of heteromerization observed for many GPCRs
and its functional relevance was not completely clear in physiological conditions
and was in some cases regarded with a degree of skepticism, but at least for the D1-D2
receptor heteromer we have shown evidence of occurrence under physiological conditions
in native tissues with emerging important functional relevance.

For D1 and D2 receptors, the presence of two anatomically segregated sets of neurons,
forming the striatonigral D1-enriched direct pathway and the striatopallidal D2-enriched
indirect pathway is commonly recognized, with D1R localizing to the dynorphin (DYN)-expressing
neurons, and D2R localizing to the enkephalin (ENK)-expressing neurons [25,26]. Recent studies emanating from fluorophore-tagged promoter elements of D1R and D2R
in bacterial artificial chromosome (BAC) transgenic mice [27] allowed an evaluation of the proportions of striatal neurons expressing D1R, D2R,
or both [28-32]. There were, however, variations in the levels of expression of EGFP between one
line and another [32], resulting in incomplete labeling of a significant proportion of striatal medium
spiny neurons (MSNs) [28]. While this method supported the segregation between the D1-enriched direct pathway
and the striatopallidal D2-enriched indirect pathway, a certain fraction of MSNs (~17%)
expressing both receptors was predicted in the NAc shell, whereas only ~5-6% of MSNs
were calculated to co-express both receptors in the dorsal striatum [30-32]. These BAC-calculated colocalization data are consistent with our data and the numerous
other reports indicating a colocalization of D1R and D2R in neurons in culture or
in situ with higher D1R and D2R co-localization observed in cultured striatal neurons
(60 to 100%) than in the adult striatum [33-40].

Presence of dopamine D1-D2 receptor heteromers in brain

Several reports indicated the presence of a D1-like receptor activating IP3 production
and/or increasing intracellular calcium in neurons in culture or slices from different
brain regions, including striatum, hippocampus, and cortex [41-44]. However, the cloned D1R was devoid of such effects when expressed in different host
cells (reviewed in 17 and 20) and persisted in a D1 receptor null mouse model [45]. We then demonstrated that dopamine D1 and D2 receptors form functional heterooligomeric
complexes in cells and in vivo [21-23,40,46] and that the mobilization of intracellular calcium was in fact a unique signaling
pathway resulting from the activation of this D1-D2 heteromeric receptor complex [21,23,40].

The presence of the D1-D2 receptor heteromer was demonstrated by different techniques
including coimmunoprecipitating both receptors from rat striatum, as well as from
cells coexpressing D1R and D2R [21,40], and by different methodologies using the fluorescence resonance energy transfer
(FRET) technique in cells [22,24], in striatal neurons [40,47] and different brain regions [40,46].

Interestingly, in adult rat brain, coexpressed dopamine D1 and D2 receptors were present
in a unique subset of neurons coexpressing both DYN and ENK neuropeptides in different
brain regions, including nucleus accumbens (NAc), caudate-putamen (CP), ventral pallidum,
globus pallidus (GP), and entopeduncular nucleus [46], with some inter-regional variation. The lowest proportion (~6-7%) of D1R-expressing
neurons that coexpress D2R was shown in the CP [40,46], whereas the highest proportion (~59%) of D1R-expressing neurons that coexpress D2R
was observed in GP [46]. A substantial number (~20-30%) of D1R neurons that coexpress D2R was also observed
in NAc [40,46], consistent with the anatomical findings resulting from BAC transgenic mice [30-32].

The direct interaction of D1R and D2R to form heteromers in brain was shown by confocal
FRET technique using two methodologies [40,46,47]. The confocal FRET technique demonstrated clearly and directly the presence of the
D1-D2 receptor heteromer in striatal neurons [40,47] and in brain in situ [40,46]. In NAc, acceptor photobleaching-based FRET showed a high FRET efficiency of ~21%
[46], in the same range (~20%) as with a second quantitative confocal FRET, that further
quantified the parameters of the interaction between D1R and D2R to calculate the
FRET efficiency and the assessment of the distance separating both fluophore-tagged
receptors [40,46]. In NAc, interactions between colocalized D1R and D2R (Figure 1) displayed high FRET efficiency (~20%) and a relative distance of 5-7 nm (50-70 Å)
(Table 1), synonymous with a close proximity between D1 and D2 receptors and indicative of
D1-D2 heteromer formation. In contrast, although an indication of D1-D2 heteromer
formation in CP was observed, the parameters, FRET efficiency (~5%) and the relative
distance of 8-9 nm (80-90 Å) between the receptors suggested that in CP either D1R-D2R
interaction was weaker, or fewer D1-D2 receptor heteromers were formed, and/or lower
order of D1-D2 oligomers than in the NAc was present [40,46].

Figure 1.Example of Confocal FRET analysis of D1 and D2 receptor interaction in a medium spiney
neuron from the core region of rat nucleus accumbens. Anti-D2-Alexa 350 (green) and anti-D1-Alexa 488 (red) were used as donor and acceptor
dipoles. The FRET signal was detected and measured in microdomains [regions of interest
(ROIs)] within the neuron coexpressing D1 and D2 receptors. Analysis shows the FRET
efficiency and the distance separating the dipoles.

The specific activation of the D1-D2 receptor heteromer in postnatal striatal neurons
[40], and from cells co-expressing D1R and D2R [21,23] resulted in the intracellular release of calcium from stores sensitive to activation
of inositol triphosphate receptors (IP3-R). This rise in intracellular calcium was
rapid, transient, independent of extracellular calcium influx, and involved the activation
of Gq protein, and phospholipase C (PLC) [21,23,40]. This calcium signal resulted in an increase in the phosphorylated-activated form
of CaMKIIα in postnatal striatal neurons [40] and rat striatum [23]. The use of dopamine D1-/-, D2-/- and D5-/- receptor null mice indicated clearly that the calcium-CaMKIIα signaling pathway exclusively
involved both D1R and D2R within a functional complex [23,40], and was different from the calcium signal generated by the activation of D5R or
the D2-D5 receptor heteromer [48,49].

Intracellular calcium plays key roles in many neuronal functions including the regulation
of synaptic transmission [50]. The intracellular calcium signaling pathway activated through the dopamine D1-D2
receptor heteromer resulted in CaMKIIα activation and BDNF production in striatal
neurons in culture as well as in the nucleus accumbens of adult rats, leading ultimately
in cultured postnatal striatal neurons to enhanced dendritic branching [40]. Both CaMKIIα and BDNF have been shown to be involved in synaptic plasticity. While
evidence has indicated that CaMKIIα is a critical regulator of synaptic plasticity
in neurons [51-54] with 50% of CaMKIIα-deficient mice presenting changes in behavior and learning [55], BDNF has been shown to modulate the branching and growth of axons, dendrites and
spines (reviewed in 56). For example, BDNF was shown to be released from cell bodies
and dendrites of cortical neurons and regulated the branching of dendrites in adjacent
neurons [57]. The BDNF effect on the dendritic morphology and also on spine morphology (reviewed
in 56) would be of great importance in the modulation of neuronal and synaptic function
and plasticity [58]. The neurotrophin signaling transduced through BDNF receptor TrkB has been recently
reported to be involved in the control of the size of the striatum by modulating the
number of medium spiny neurons (MSNs), with deletion of the gene for the TrkB receptor
in striatal progenitors leading to the loss of almost 50% of MSNs without affecting
striatal interneurons [59]. Also, the BDNF signaling through TrkB was shown to be involved in the induction
and the maintenance of synaptic plasticity, through its long-term potentiation (LTP)
component [60]. The other component, long-term depression (LTD) was shown to involve BDNF signaling
through the receptor p75 in hippocampal slices from p75-deficient mice [61]. BDNF plays also an important role in the modulation of neurotransmitter release,
a key step in synaptic plasticity [56]. The release of glutamate for example involves PLC and BDNF through a mechanism involving
a rise in intracellular calcium via a release from IP3 receptor-sensitive stores [62,63]. It is very interesting to draw the parallel between these mechanisms by which CaMKII
and BDNF modulate synaptic plasticity and the signaling pathway revealed with the
activation of dopamine D1-D2 receptor heteromer in the striatum [40], which also involves PLC, the intracellular calcium release from IP3 receptor-sensitive
stores, CaMKII activation and BDNF production. This suggests that the D1-D2 receptor
heteromer-mediated signaling pathway may play an essential role in synaptic plasticity,
notably in its LTP component [20,40,49], the dysregulation of which may lead to alterations in cognition, learning, and memory
that contribute to the pathophysiology of dopamine-related disorders such as schizophrenia
or drug addiction [20,40,46,49].

Further, we showed that in rat striatum amphetamine administration significantly increased
the affinity of SKF 83959, a specific D1-D2 receptor heteromer agonist [64], by 10-fold for the D1-D2 receptor heteromer and increased the proportion of the
D1-D2 heteromer in the agonist-detected high affinity state [46]. GTPγS binding studies indicated that the D1-D2 heteromer was functionally supersensitive
in response to repeated increases in dopamine transmission following amphetamine administration
[46]. In addition to increasing the activity and sensitivity of D1-D2 receptor heteromers,
amphetamine also increased the D1-D2 receptor heteromer density in the NAc as assessed
by FRET technique [46].

Interestingly, the increase in the proportion of D1-D2 heteromers in the high affinity
state was also detected in schizophrenia globus pallidus (GP) [46]. Amphetamine treatment leading to increased dopamine transmission and behavioral
sensitization has been used as an animal model for schizophrenia [65], since schizophrenia has been linked to increased dopamine transmission [66]. Moreover, the different components of calcium signaling, including Gq proteins,
PLC, and CaMKII were shown to be affected in the brains of schizophrenia patients
[67]. Given these facts, the findings showing an increase in the proportion of D1-D2 heteromers
in high affinity state in both schizophrenia and chronic amphetamine treatment may
indicate a preponderant role of the D1-D2 receptor heteromer-mediated calcium-CaMKII-BDNF
signaling pathway in both drug addiction and schizophrenia.

This D1-D2 receptor heteromer-calcium signal may represent a first common biochemical
bridge between the dopaminergic system-CaMKII-BDNF, synaptic plasticity and the occurrence
of drug addiction and schizophrenia. The finding that the activation of CaMKIIα was
necessary for the induction of behavioral sensitization to drugs [68], a physiological phenomenon that also requires the coactivation of D1 and D2 dopamine
receptors [14], provides additional evidence of the important role of dopamine D1-D2 receptor heteromer-calcium
signal in drug addiction.

After years of some skepticism surrounding the physiological presence and relevance
of GPCR homo- and hetero-oligomers, there is ample evidence for the presence in the
brain of a unique entity, the D1-D2 receptor heteromer, with a unique signaling pathway
different from the signals generated by each receptor homomer, with a physiological
relevance and high importance in at least two major pathologies, schizophrenia and
drug addiction, making the D1-D2 receptor an interesting therapeutic target for these
disorders.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

All authors read and approved the final manuscript

Acknowledgements

One of a series of four reviews on G protein-coupled receptors published in memory
of Hubert H. M. Van Tol (1959-2006), formerly Head of Molecular Biology at the Centre
for Addiction and Mental Health, and a Professor in the Departments of Psychiatry
and Pharmacology at the University of Toronto. Hubert's contributions to G protein-coupled
receptor research and neuroscience are numerous and are best remembered by his central
role in the cloning of the dopamine receptor family. His many achievements were recognized
through awards such as the John Dewan award, The Prix Galien, and the Joey & Toby
Tanenbaum Distinguished Scientist Award for Schizophrenia Research.

SRG is the holder of a Tier 1 Canada Research Chair in Molecular Neuroscience. This
work was supported by a grant from the NIH National Institute of Drug Abuse.

Braun AR, Laruelle M, Mouradian MM: Interactions between D1 and D2 dopamine receptor family agonists and antagonists:
the effects of chronic exposure on behavior and receptor binding in rats and their
clinical implications.